BI203 Lecture 17: Protein Sorting and Transport - Slides 1-20 (Import into the ER)

Question 1

Subdivision of the _____ allows these cells to function efficiently in spite of their large size — at least a thousand times the volume of bacteria.

Answer 1

cytoplasm

Question 2

The endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes are involved in protein processing and are connected by ____ transport.

Answer 2

vesicular

Question 3

The ____ ____, ____ ____, ____, and ____ are involved in protein processing and are connected by vesicular transport.

Answer 3

Endoplasmic reticulum (ER), golgi apparatus, endosomes, lysosomes

Question 4

The ____ ____ is a network of membrane-enclosed tubules and sacs (cisternae), extending from the nuclear membrane throughout the cytoplasm.

The membrane is continuous and is the largest organelle of most eukaryotic cells.

Answer 4

endoplasmic reticulum (ER)

Question 5

The endoplasmic reticulum (ER) is a network of membrane-enclosed tubules and sacs (____), extending from the nuclear membrane throughout the cytoplasm.

The membrane is continuous and is the largest organelle of most eukaryotic cells.

Answer 5

cisternae

Question 6

The ER has two domains that perform different functions:
____ ____ - is covered by ribosomes on the outer surface.
____ ____ - has no ribosomes and is involved in lipid metabolism.

Answer 6

Rough ER, Smooth ER

Question 7

Proteins destined for many compartments are imported following translation on ____ ____.

Answer 7

free ribosomes

Question 8

Proteins are trafficked (or sorted) to their respective compartments via the ___ ___.

Answer 8

secretory pathway

Question 9

Key Study: Examined the path through which ___ ___ ___ are secreted from cells from pancreatic cells.

Answer 9

newly synthesized proteins

Question 10

Key Study Method:

1) ___ newly synthesized proteins with ___ amino acids.
2) Chased the proteins with ___ amino acids.
3) Tracked the location of the radiolabeled protein through ___.

Answer 10

pulse-labeled, hot, cold, time

Question 11

The Secretory Pathway: ___ ___ -> ___ ___ -> ___ ___ -> ___ ___.

Answer 11

ER (ER) -> Golgi Bodies -> Secretory Vesicles -> Cell Exterior

Question 12

The role of the ER in protein processing and sorting was first demonstrated by Palade and colleagues in the 1960s.

They studied ___ ___ cells that secrete digestive enzymes into the small intestine.

Newly synthesized proteins were labeled with radioisotopes.

Location of the radiolabeled proteins was then determined by ___.

After labeling, incubation with non-labeled amino acids for different lengths of time (called a “___”) allowed them to track the labeled proteins through the ER, Golgi apparatus, secretory vesicles, and then outside the cell.

Answer 12

pancreatic acinar cells, autoradiography, “chase”

Question 13

In eukaryotic cells, initial sorting takes place while translation is in progress.

Proteins synthesized on free ribosomes stay in the ___ or are transported to the nucleus and other organelles.

Proteins synthesized on membrane-bound ribosomes are translocated directly into the ___ ___.

Answer 13

cytosol, Endoplasmic Reticulum (ER)

Question 14

Ribosomes are targeted to the ER by a ___ ___ at the amino terminus.

The signal is removed when the growing polypeptide chain enters the ER.

Answer 14

signal sequence

Question 15

Amino acid sequences at the N-terminus of a growing polypeptide chain targets the ribosome to the ER -> ___ ___.

Answer 15

signal sequence

Question 16

KNOWN:

1) Translation of secreted proteins takes place on ER-bound ribosomes.
2) The protein was transferred across the membrane during synthesis.
3) Secreted protein had ___ amino acids missing at its N-terminus compared to protein translated in vitro on “free ribosomes”.

Answer 16

20

Question 17

The role of signal sequences was determined by ___ ___ preparations of the rough ER.

Answer 17

in vitro

Question 18

When cells are disrupted, the ER breaks up into small vesicles called ___.

Answer 18

microsomes

Question 19

Rough microsomes were more dense (___) than smooth microsomes.

Answer 19

heavier

Question 20

Blobel and Sabatini (1975) found that translation of secretory proteins on free ribosomes retained the signal sequences and were slightly ___.

When microsomes were added, the growing polypeptides were ___ into the microsomes and the signal sequences were ___.

Answer 20

larger, incorporated, removed

Question 21

The protein was incorporated into the microsomes and was ___ than the lgG translated with microsomes.

Answer 21

larger

Question 22

Polypeptides translated on free ribosomes can be targeted to the ___, ___, ___, or ___ via their signal sequences.

Answer 22

nucleus, mitochondria, chloroplasts, peroxisomes

Question 23

Polypeptides translated on membrane-bound ribosomes can be targeted to the ___ ___, ___ ___, ___, or ___ via their signal sequences.

Answer 23

plasma membrane, secretory vesicles, endosomes, lysosomes

Question 24

Signal sequences (about 20 amino acids) include a stretch of ___ residues, and are usually located at the amino terminus of the polypeptide chain.

Answer 24

hydrophobic

Question 25

Once the polypeptide reaches the ER:

Insertion of the signal sequence opens the ___ by moving a plug away from the channel.

Elongation of the polypeptide drives its transfer through the ___.

Answer 25

translocon

Question 26

As they emerge from the ribosome, signal sequences are bound by a ___ ___ ___ (SRP).

Answer 26

signal recognition particle (SRP)

Question 27

SRPs consist of ___ polypeptides and a small cytoplasmic RNA (SRP RNA).

Answer 27

6

Question 28

The SRP binds the ribosome and the signal sequence, inhibiting further translation.

The entire complex binds to a ___ ___ on the rough ER membrane.

Answer 28

SRP receptor

Question 29

The SRP is then released, and the ribosome binds to a membrane channel or ___.
The signal sequence is inserted into the translocon, and translation resumes.
The signal sequence is cleaved by ___ ___ and released into the ER lumen.

Answer 29

translocon, signal peptidase

Question 30

More recent research has shown that some proteins are targeted to the ER post-translationally via interactions b/w the signal sequence & the ___ ___.

Answer 30

Sec62/63 complex

Question 31

___ = also a Hsp70 chaperone that facilitates folding of proteins following translocation into the ER.

Answer 31

BiP

Question 32

In ___ translocation (more common in yeast), polypeptides are targeted to the ER when translation is complete.

The signal sequences are recognized by different receptors on the translocon.

Hsp70 and Hsp40 chaperones keep the polypeptide chains unfolded so they can enter the translocon.
Another Hsp70 chaperone in the ER (BiP) acts as a ___ to pull the polypeptide chain through the channel and into the ER.

Answer 32

post-translation, ratchet

Question 33

Point: The primary role of lumenal ER ___ is to assist in the proper folding and assembly on nascent polypeptides.

Answer 33

proteins